Tuesday, 23 April 2013

Understanding exactly how stem cells form into specific organs and tissues is the holy grail of regenerative medicine. Now a UC Santa Barbara researcher has added to that body of knowledge by determining how stem cells produce different types of "daughter" cells in Drosophila (fruit flies). The findings appear today in the Proceedings of the National Academy of Sciences.

Denise Montell, Duggan Professor of Molecular, Cellular and Developmental Biology at UCSB, and colleagues studied the ovaries of fruit flies in order to see stem cells in their natural environment. Because these organisms are excellent models for understanding stem cell biology, researchers were able to shed light on the earliest stages of follicle cell differentiation, a previously poorly understood area of developmental biology.

"It is clear that the fundamental principles that control cell behaviour in simple animals are conserved and control the behaviour of our cells as well," she said.

"There is so much we can learn by studying simple organisms."

This
is a schematic drawing of a

Drosophila ovariole
and a magnified

germanium.
Credit: UCSB.

Using a nuclear protein expressed in follicle stem cells (FSCs), the researchers found that castor, which plays an important role in specifying which types of brain cells are produced during embryonic development, also helps maintain FSCs throughout the life of the animal.

"Having identified this important protein molecule in fruit flies, we can test whether the human version of the protein is important for stem cells and their daughters as well," said Montell.

"The more we know about the molecules that govern stem cell behaviour, the closer we will get to control these cells."

This image shows Denise Montell,

University of California, Santa
Barbara.

Credit: George Foulsham, UCSB.

Her research team placed the evolutionarily conserved castor (Cas) gene, which encodes a zinc finger protein, in a genetic circuit with two other evolutionarily conserved genes, hedgehog (Hh) and eyes absent (Eya), to determine the fates of specific cell progeny (daughters). What's more, they identified Cas as a critical, tissue-specific target of Hh signalling, which not only plays a key role in maintaining follicle stem cells but also assists in the diversification of their progeny.

The study also shows that complementary patterns of Cas and Eya reveal the gradual differentiation of polar and stalk precursor cells at the earliest stages of their development. In addition, it provides a marker for cell fates and insight into the molecular and cellular mechanisms by which FSC progeny diverge into distinct fates.

Follicle cells undergo a binary choice during early differentiation. Those that turn into specialized cells found at the poles of egg chambers go on to make two cell types: polar and stalk. The three genes, Cas, Eya and Hh, work in various combinations, sometimes repressively, to determine which types of cells are formed. Cas is required for polar and stalk cell fate specification, while Eya is a negative regulator of these cells' fate. Hh is necessary for Cas to be expressed, and Hh signalling is essential to repress Eya.

"If you just had one of these markers, it was hard to tell what's going on," explained Montell.

"All the cells looked the same and you had no idea when or how the process occurred. But now we can actually see how the cells acquire different identities."

Hh also plays many roles in embryonic development, adult homeostasis, birth defects, and cancer. Hh antagonists are currently in clinical trials for the treatment of several types of cancer. However, Hh signalling is important in so many different cell types and tissues that systemic delivery of such inhibitors may cause serious side effects. Therefore identifying the essential, tissue-specific effectors of Hh has the potential to lead to the identification of more specific therapeutic targets.

Someday, targeted inhibition of Hh signalling may be effective in the treatment and prevention of many types of human cancers.

In a serendipitous discovery, scientists at The Scripps Research Institute (TSRI) have found a way to turn bone marrow stem cells directly into brain cells.

Current techniques for turning patients' marrow cells into cells of some other desired type are relatively cumbersome, risky and effectively confined to the lab dish. The new finding points to the possibility of simpler and safer techniques. Cell therapies derived from patients' own cells are widely expected to be useful in treating spinal cord injuries, strokes and other conditions throughout the body, with little or no risk of immune rejection.

Scientists at the Scripps Research
Institute

have found a simple way to turn bone
marrow

stem cells directly into brain
precursor cells,

such as those shown here. Credit: Image

courtesy of the Lerner lab, The Scripps
Research

Institute.

"These results highlight the potential of antibodies as versatile manipulators of cellular functions," said Richard A. Lerner, the Lita Annenberg Hazen Professor of Immunochemistry and institute professor in the Department of Cell and Molecular Biology at TSRI, and principal investigator for the new study.

"This is a far cry from the way antibodies used to be thought of — as molecules that were selected simply for binding and not function."

The researchers discovered the method, reported in the online Early Edition of the Proceedings of the National Academy of Sciences the week of April 22, 2013, while looking for lab-grown antibodies that can activate a growth-stimulating receptor on bone marrow cells. One antibody turned out to activate the receptor in a way that induces marrow stem cells — which normally develop into white blood cells — to become neural progenitor cells, a type of almost-mature brain cell.

Nature's Toolkit

Natural antibodies are large, Y-shaped proteins produced by immune cells. Collectively, they are diverse enough to recognize about 100 billion distinct shapes on viruses, bacteria and other targets. Since the 1980s, molecular biologists have known how to produce antibodies in cell cultures in the laboratory. That has allowed them to start using this vast, target-gripping toolkit to make scientific probes, as well as diagnostics and therapies for cancer, arthritis, transplant rejection, viral infections and other diseases.

In the late 1980s, Lerner and his TSRI colleagues helped invent the first techniques for generating large "libraries" of distinct antibodies and swiftly determining which of these could bind to a desired target. The anti-inflammatory antibody Humira®, now one of the world's top-selling drugs, was discovered with the benefit of this technology.

Hongkai Zhang, research associate in
the

Lerner lab, which discovered a method
for

rapidly finding antibodies that have a
desired

effect on cells, not just a desired
ability to

bind to a target. Credit: Photo
by Cindy

Brauer.

Last year, in a study spearheaded by TSRI Research Associate Hongkai Zhang, Lerner's laboratory devised a new antibody-discovery technique — in which antibodies is produced in mammalian cells along with receptors or other target molecules of interest. The technique enables researchers to determine rapidly not just which antibodies in a library bind to a given receptor, for example, but also which ones activate the receptor and thereby alter cell function.

Lab Dish in a Cell

For the new study, Lerner laboratory Research Associate Jia Xie and colleagues modified the new technique so that antibody proteins produced in a given cell are physically anchored to the cell's outer membrane, near its target receptors.

"Confining an antibody's activity to the cell in which it is produced effectively allows us to use larger antibody libraries and to screen these antibodies more quickly for a specific activity," said Xie. With the improved technique, scientists can sift through a library of tens of millions of antibodies in a few days.

In an early test, Xie used the new method to screen for antibodies that could activate the GCSF receptor, a growth-factor receptor found on bone marrow cells and other cell types. GCSF-mimicking drugs were among the first biotech bestsellers because of their ability to stimulate white blood cell growth — which counteracts the marrow-suppressing side effect of cancer chemotherapy.

The team soon isolated one antibody type or "clone" that could activate the GCSF receptor and stimulate growth in test cells. The researchers then tested an unanchored, soluble version of this antibody on cultures of bone marrow stem cells from human volunteers. Whereas the GCSF protein, as expected, stimulated such stem cells to proliferate and start maturing towards adult white blood cells, the GCSF-mimicking antibody had a markedly different effect.

"The cells proliferated, but also started becoming long and thin and attaching to the bottom of the dish," remembered Xie.

To Lerner, the cells were reminiscent of neural progenitor cells — which further tests for neural cell markers confirmed they were.

A New Direction

Changing cells of marrow lineage into cells of neural lineage — a direct identity switch termed "transdifferentiation" — just by activating a single receptor is a noteworthy achievement. Scientists do have methods for turning marrow stem cells into other adult cell types, but these methods typically require a radical and risky deprogramming of marrow cells to an embryonic-like stem-cell state, followed by a complex series of molecular nudges toward a given adult cell fate. Relatively few laboratories have reported direct transdifferentiation techniques.

"As far as I know, no one has ever achieved transdifferentiation by using a single protein — a protein that potentially could be used as a therapeutic," said Lerner.

Current cell-therapy methods typically assume that a patient's cells will be harvested, then reprogrammed and multiplied in a lab dish before being re-introduced into the patient. In principle, according to Lerner, an antibody such as the one they have discovered could be injected directly into the bloodstream of a sick patient. From the bloodstream it would find its way to the marrow, and, for example, convert some marrow stem cells into neural progenitor cells.

"Those neural progenitors would infiltrate the brain, find areas of damage and help repair them," he said.

While the researchers still aren't sure why the new antibody has such an odd effect on the GCSF receptor, they suspect it binds the receptor for longer than the natural GCSF protein can achieve, and this lengthier interaction alters the receptor's signalling pattern. Drug-development researchers are increasingly recognizing that subtle differences in the way a cell-surface receptor is bound and activated can result in very different biological effects. That adds complexity to their task, but in principle expands the scope of what they can achieve.

"If you can use the same receptor in different ways, then the potential of the genome is bigger," said Lerner.

Thursday, 18 April 2013

Using a new stem-cell based drug screening technology with the potential to reinvent and greatly reduce the cost of the way new pharmaceuticals are developed, Harvard Stem Cell Institute (HSCI) researchers have found a compound more effective in protecting the neurons killed in amyotrophic lateral sclerosis (ALS) – Lou Gehrig's disease – than two drugs that failed in human clinical trials after hundreds of millions of dollars had been invested in them.

"It's a deep, dark secret of drug discovery that very few drugs have been tested on human-diseased cells before being tested in a live person," said Rubin, who heads HSCI's program in translational medicine.

"We were interested in the notion that we can use stem cells to correct that situation."

Rubin's model is built on an earlier proof-of-concept developed by HSCI Principal Faculty member Kevin Eggan, who demonstrated that it was possible to move a neuron-based disease into a laboratory dish using stem cells carrying the genes of patients with the disease.

The c-Jun-mediated cell death pathway
(marked

by red nuclei that are positive for
phospho-cjun) is

activated in stem cell-derived motor
neurons (green)

exposed to trophic factor withdrawal
(upper left panel).

C-Jun activation and cell death are
blocked by

kenpaullone, an inhibitor GSK-3 and HGK
(MAP4K4)

kinases (lower right panel; kenpaullone
structure

superimposed). Credit:Cell Stem
Cell, Yang et al..

In a paper published today in the journal Cell Stem Cell, Rubin lays out how he and his colleagues applied their new method of stem cell-based drug discovery to ALS. The disease is associated with the progressive death of motor neurons, which pass information between the brain and the muscles. As cells die, people with ALS experience weakness in their limbs followed by rapid paralysis and respiratory failure. The disease typically strikes later in life. Ten percent of cases are genetically predisposed, but for most patients there is no known trigger.

Rubin's lab began by first studying the disease in mice, growing billions of motor neurons from mouse embryonic stem cells, half normal and half with a genetic mutation known to cause ALS.

Investigators starved the cells of nutrients and then screened five thousand drug-like molecules to find any that would keep the motor neurons alive.

Several hits were identified, but the molecule that best prolonged the life of both normal and ALS motor neurons was kenpaullone, previously known for blocking the action of an enzyme (GSK-3) that switches on and off several cellular processes, including cell growth and death.

Kenpaullone proved effective in several follow-up experiments that put mouse motor neurons in situations of certain death. Neuron survival increased in the presence of the molecule whether the cells were programmed to die or placed in a toxic environment.

After further investigation, Rubin's lab discovered kenpaullone's potency comes from its ability to also inhibit HGK – an enzyme that sets off a chain of reactions that leads to motor neuron death. This enzyme was not previously known to be important in motor neurons or associated with ALS, marking the discovery of a new drug target for the disease.

"I think that stem-cell screens will discover new compounds that have never been discovered before by other methods," Rubin said.

"I'm excited to think that someday one of them might actually be good enough to go into the clinic."

To find out if kenpaullone works in diseased human cells, Rubin's lab exposed patient motor neurons and motor neurons grown from human embryonic stem cells to the molecule, as well as two drugs that did well in mice but failed in phase III human clinical trials for ALS. Once again, kenpaullone increased the rate of neuron survival, while one drug saw little response, and the other drug failed to keep any cells alive.

According to Rubin, before kenpaullone could be used as a drug, it would need a substantial molecular makeover to make it better able to target cells and find its way into the spinal cord so it can access motor neurons.

"This is kind of a proof of principle on the do-ability of the whole thing," he said.

"I think it's possible to use this method to discover new drug targets and to pre-validate compounds on real human disease cells before putting them in the clinic."

In the meantime, Rubin's next steps will be to continue searching for better drug-like compounds that can inhibit HGK and thus enhance motor neuron survival. He believes that the new information that comes out of this research will be useful to academia and the pharmaceutical industry.

"These kinds of exploratory screens are hard to fund, so being part of the HSCI" – which provided some of the funding – "has been absolutely essential," Rubin said.

Wednesday, 17 April 2013

Stem cells and tissue-specific cells
can be grown in abundance from mature mammalian cells simply by blocking a
certain membrane protein, according to scientists at the University
of Pittsburgh School of Medicine and the National
Institutes of Health (NIH). Their experiments, reported today in Scientific
Reports, also show that the process doesn't require other kinds of cells or
agents to artificially support cell growth and doesn't activate cancer genes.

Scientists hope that lab-grown stem cells and induced pluripotent stem (iPS) cells, which have the ability to produce specialized cells such as neurons and cardiac cells, could one day be used to treat diseases and repair damaged tissues, said co-author Jeffrey S. Isenberg, M.D., associate professor, Division of Pulmonary, Allergy and Critical Care Medicine, Pitt School of Medicine.

"Even though stem cells are able to self-renew, they are quite challenging to grow in the lab," he said.

"Often you have to use feeder cells or introduce viral vectors to artificially create the conditions needed for these cells to survive and thrive."

In 2008, prior to joining Pitt, Dr. Isenberg was working in the National Cancer Institute (NCI) lab of senior author David D. Roberts, Ph.D., using agents that block a membrane protein called CD47 to explore their effects on blood vessels. He noticed that when cells from the lining of the lungs, called endothelium, had been treated with a CD47 blocker, they stayed healthy and maintained their growth and function for months.

Dr. Roberts' NIH team continued to experiment with CD47 blockade, focusing on defining the underlying molecular mechanisms that control cell growth.

They found that endothelial cells obtained from mice lacking CD47 multiplied readily and thrived in a culture dish, unlike those from control mice. Lead author Sukhbir Kaur, Ph.D., discovered that this resulted from increased expression of four genes that are regarded to be essential for formation of iPS cells. When placed into a defined growth medium, cells lacking CD47 spontaneously formed clusters characteristic of iPS cells. By then introducing various growth factors into the culture medium, these cells could be directed to become cells of other tissue types. Despite their vigorous growth, they didn't form tumours when injected into mice, a major disadvantage when using existing iPS cells.

"Stem cells prepared by this new procedure should be much safer to use in patients," Dr. Roberts noted.

"Also, the technique opens up opportunities to treat various illnesses by injecting a drug that stimulates patients to make more of their own stem cells."

"These experiments indicate that we can take a primary human or other mammalian cell, even a mature adult cell, and by targeting CD47 turn on its pluripotent capability,” according to Dr. Isenberg.

We can get brain cells, liver cells, muscle cells and more. In the short term, they could be a boon for a variety of research questions in the lab."

In the future, blocking CD47 might make it possible to generate large numbers of healthy cells for therapies, such as alternatives to conventional bone marrow transplantation and complex tissue and organ bioengineering, he added.

"These
exciting findings provide a rationale for using CD47 blocking therapies to
increase stem cell uptake and survival in transplanted organs, matrix grafts,
or other applications," said Mark Gladwin,
M.D., professor and chief, Division of Pulmonary, Allergy and Critical Care
Medicine, Pitt School of Medicine.

"This continues a strong and productive collaboration between investigators at the NCI and the University of Pittsburgh's Vascular Medicine Institute."

Sunday, 14 April 2013

Scientists at CWRU School of Medicine discover new technique that holds promise for the treatment of multiple sclerosis and cerebral palsy

Sunday, 14 April 2013

Researchers at Case Western Reserve School of Medicine have discovered a technique that directly converts skin cells to the type of brain cells destroyed in patients with multiple sclerosis, cerebral palsy and other so-called myelin disorders.

This discovery appears today in the journal Nature Biotechnology.

This breakthrough now enables "on demand" production of myelinating cells, which provide a vital sheath of insulation that protects neurons and enables the delivery of brain impulses to the rest of the body. In patients with multiple sclerosis (MS), cerebral palsy (CP), and rare genetic disorders called leukodystrophies, myelinating cells are destroyed and cannot be replaced.

The new technique involves directly converting fibroblasts — an abundant structural cell present in the skin and most organs — into oligodendrocytes, the type of cell responsible for myelinating the neurons of the brain.

"Its 'cellular alchemy,'" explained Paul Tesar, PhD, assistant professor of genetics and genome sciences at Case Western Reserve School of Medicine and senior author of the study.

"We are taking a readily accessible and abundant cell and completely switching its identity to become a highly valuable cell for therapy."

In a process termed "cellular reprogramming," researchers manipulated the levels of three naturally occurring proteins to induce fibroblast cells to become precursors to oligodendrocytes (called oligodendrocyte progenitor cells, or OPCs).

Tesar's team, led by Case Western Reserve researchers and co-first authors Fadi Najm and Angela Lager, rapidly generated billions of these induced OPCs (called iOPCs). Even more important, they showed that iOPCs could regenerate new myelin coatings around nerves after being transplanted to mice — a result that offers hope the technique might be used to treat human myelin disorders.

When oligodendrocytes are damaged or become dysfunctional in myelinating diseases, the insulating myelin coating that normally coats nerves is lost. A cure requires the myelin coating to be regenerated by replacement oligodendrocytes.

Until now, OPCs and oligodendrocytes could only be obtained from foetal tissue or pluripotent stem cells. These techniques have been valuable, but with limitations.

"The myelin repair field has been hampered by an inability to rapidly generate safe and effective sources of functional oligodendrocytes," explained co-author and myelin expert Robert Miller, PhD, professor of neurosciences at the Case Western Reserve School of Medicine and the university's vice president for research.

"The new technique may overcome all of these issues by providing a rapid and streamlined way to directly generate functional myelin producing cells."

This initial study used mouse cells. The critical next step is to demonstrate feasibility and safety using human cells in a lab setting. If successful, the technique could have widespread therapeutic application to human myelin disorders.

"The progression of stem cell biology is providing opportunities for clinical translation that a decade ago would not have been possible," said Stanton Gerson, MD, professor of Medicine-Hematology/Oncology at the School of Medicine and director of the National Center for Regenerative Medicine and the UH Case Medical Center Seidman Cancer Center.

Bioengineered rat kidneys developed by Massachusetts General Hospital (MGH) investigators successfully produced urine both in a laboratory apparatus and after being transplanted into living animals. In their report, receiving advance online publication in Nature Medicine, the research team describes building functional replacement kidneys on the structure of donor organs from which living cells had been stripped, an approach previously used to create bioartificial hearts, lungs and livers.

Removal
of all living cells from a rat kidney leaves
a collagen scaffolding, ready for repopulation with
new kidney and vascular cells. Credit: Massachusetts
General Hospital Center for Regenerative Medicine.

"What is unique about this approach is that the native organ's architecture is preserved, so that the resulting graft can be transplanted just like a donor kidney and connected to the recipient's vascular and urinary systems," says Harald Ott, MD, PhD, of the MGH Center for Regenerative Medicine, senior author of the Nature Medicine article.

"If this technology can be scaled to human-sized grafts, patients suffering from renal failure who are currently waiting for donor kidneys or who are not transplant candidates could theoretically receive new organs derived from their own cells."

Around 18,000 kidney transplants are performed in the U.S. each year, but 100,000 Americans with end-stage kidney disease are still waiting for a donor organ. Even those fortunate enough to receive a transplant face a lifetime of immunosuppressive drugs, which pose many health risks and cannot totally eliminate the incidence of eventual organ rejection.

This is
a previously decellularized rat kidney after
reseeding with endothelial cells, to repopulate the
organ's vascular system, and neonatal kidney cells. Credit: Massachusetts General Hospital Center
for Regenerative Medicine.

The approach used in this study to engineer donor organs, based on a technology that Ott discovered as a research fellow at the University of Minnesota, involves stripping the living cells from a donor organ with a detergent solution and then repopulating the collagen scaffold that remains with the appropriate cell type – in this instance human endothelial cells to replace the lining of the vascular system and kidney cells from new-born rats. The research team first decellularized rat kidneys to confirm that the organ's complex structures would be preserved. They also showed the technique worked on a larger scale by stripping cells from pig and human kidneys.

Making sure the appropriate cells were seeded into the correct portions of the collagen scaffold required delivering vascular cells through the renal artery and kidney cells through the ureter. Precisely adjusting the pressures of the solutions enabled the cells to be dispersed throughout the whole organs, which were then cultured in a bioreactor for up to 12 days. The researchers first tested the repopulated organs in a device that passed blood through its vascular system and drained off any urine, which revealed evidence of limited filtering of blood, molecular activity and urine production.

Bioengineered kidneys transplanted into living rats from which one kidney had been removed began producing urine as soon as the blood supply was restored, with no evidence of bleeding or clot formation. The overall function of the regenerated organs was significantly reduced compared with that of normal, healthy kidneys, something the researchers believe may be attributed to the immaturity of the neonatal cells used to repopulate the scaffolding.

"Further refinement of the cell types used for seeding and additional maturation in culture may allow us to achieve a more functional organ," says Ott.

"Based on this initial proof of principle, we hope that bioengineered kidneys will someday be able to fully replace kidney function just as donor kidneys do. In an ideal world, such grafts could be produced 'on demand’ from a patient's own cells, helping us overcome both the organ shortage and the need for chronic immunosuppression. We're now investigating methods of deriving the necessary cell types from patient-derived cells and refining the cell-seeding and organ culture methods to handle human-sized organs."

Ott's team focuses on the regeneration of hearts, lungs, kidneys and grafts made of composite tissues, while other teams – including one from the MGH Center for Engineering in Medicine – are using the decellularization technique to develop replacement livers

Friday, 12 April 2013

CWRU research developing technique with promise to guide formation of complex tissues

Friday, 12 April 2013

Stem cells can be coaxed to grow into new bone or new cartilage better and faster when given the right molecular cues and room inside a water-loving gel, researchers at Case Western Reserve University show.

By creating a three-dimensional checkerboard — one with alternating highly connected and less connected spaces within the hydrogel — the team found adjusting the size of the micro-pattern could affect stem cell behaviours, such as proliferation and differentiation.

Inducing how and where stem cells grow — and into the right kind of cell in three dimensions — has proven a challenge to creating useful stem cell therapies. This technique holds promise for studying how physical, chemical and other influences affect cell behaviour in three-dimensions, and, ultimately, as a method to grow tissues for regenerative medicine applications.

"We think that control over local biomaterial properties may allow us to guide the formation of complex tissues," said Eben Alsberg, an associate professor of Biomedical Engineering at Case Western Reserve.

"With this system, we can regulate cell proliferation and cell-specific differentiation into, for example, bone-like or cartilage-like cells."

Oju Jeon, PhD, a postdoctoral researcher in Biomedical Engineering, pursued this work with Alsberg. Their work is described April 11, 2013 in the online edition of Advanced Functional Materials.

Hydrogels are hydrophilic three-dimensional networks of water-soluble polymers bonded, or cross-linked, to one another. Crosslinks increase rigidity and alter the porous structure inside the gel.

Alsberg and Jeon used a hydrogel of oxidized methacrylated alginate and an 8-arm poly(ethylene glycol) amine. A chemical reaction between the alginate and the poly(ethylene glycol) creates crosslinks that provide structure within the gel.

They tweaked the mix so that a second set of crosslinks forms when exposed to light. They used checkerboard masks to create patterns of alternating singly and doubly cross-linked spaces.

The spaces, which varied in size at 25, 50, 100 and 200 micrometres across, were evenly singly and doubly cross-linked.

Human stem cells isolated from fat tissue were encapsulated in the singly and doubly cross-linked regions. The doubly cross-linked spaces are comparatively cluttered with structures. The cells grew into clusters in the singly cross-linked regions, but remained mostly isolated in the doubly cross-linked regions.

The larger the spaces in the checkerboard, the larger the clusters grew.

Cells were cultivated in media that promote differentiation into either bone or cartilage.

In both the singly and doubly cross-linked spaces, stem cells increasingly differentiated according to the media composition as the space size increased. The results were more dramatic in the singly cross-linked spaces.

"Potentially, what's happening is the single cross-linked regions allow better nutrient transport and provide more space for cells to interact and, because it's less restrictive, there's space for new cells and matrix production," Alsberg said.

"Cluster formation, in turn, may influence proliferation and differentiation. Differences in mechanical properties between regions likely also regulate the cell behaviours."

The researchers are continuing to use micro-patterning to understand the influences of biomaterials on stem cell fate decisions. This approach may permit local control over cell behaviour and, ultimately, allow the engineering of complex tissues comprised of multiple cell types using a single stem cell source.

Thursday, 11 April 2013

Years of mouse research lead to discovery of how autophagy keeps neural stem cells ready to replace damaged brain and nerve cells

Thursday, 11 April 2013

Deep inside your brain, a legion of stem cells lies ready to turn into new brain and nerve cells whenever and wherever you need them most. While they wait, they keep themselves in a state of perpetual readiness – poised to become any type of nerve cell you might need as your cells age or get damaged.

Now, new research from scientists at the University of Michigan Medical School reveals a key way they do this: through a type of internal "spring cleaning" that both clears out garbage within the cells, and keeps them in their stem-cell state.

These
colourful images show the importance
of the FIP200 gene to neural stem cell activity
and ability to become any type of brain or nerve
cell. The second column of images is from mice
that lack FIP200, compared with normal mice
(first column) and mice that lacked the p53 gene
in addition FIP200 third column) or alone (fourth
column). Credit: Guan
Laboratory, University
of Michigan.

In a paper published online in Nature Neuroscience, the U-M team shows that a particular protein, called FIP200, governs this cleaning process in neural stem cells in mice. Without FIP200, these crucial stem cells suffer damage from their own waste products – and their ability to turn into other types of cells diminishes.

It is the first time that this cellular self-cleaning process, called autophagy, has been shown to be important to neural stem cells.

The findings may help explain why aging brains and nervous systems are more prone to disease or permanent damage, as a slowing rate of self-cleaning autophagy hampers the body's ability to deploy stem cells to replace damaged or diseased cells. If the findings translate from mice to humans, the research could open up new avenues to prevention or treatment of neurological conditions.

In a related review article just published online in the journal Autophagy, the lead U-M scientist and colleagues from around the world discuss the growing evidence that autophagy is crucial to many types of tissue stem cells and embryonic stem cells as well as cancer stem cells.

As stem cell-based treatments continue to develop, the authors say, it will be increasingly important to understand the role of autophagy in preserving stem cells' health and ability to become different types of cells.

"The process of generating new neurons from neural stem cells, and the importance of that process, is pretty well understood, but the mechanism at the molecular level has not been clear," says Jun-Lin Guan, Ph.D., the senior author of the FIP200 paper and the organizing author of the autophagy and stem cells review article.

"Here, we show that autophagy is crucial for maintenance of neural stem cells and differentiation, and show the mechanism by which it happens."

Through autophagy, he says, neural stem cells can regulate levels of reactive oxygen species – sometimes known as free radicals – that can build up in the low-oxygen environment of the brain regions where neural stem cells reside. Abnormally higher levels of ROS can cause neural stem cells to start differentiating.

The new discovery, made after 15 years of research with funding from the National Institutes of Health, shows the importance of investment in lab science – and the role of serendipity in research.

Guan has been studying the role of FIP200 – whose full name is focal adhesion kinase family interacting protein of 200 kD – in cellular biology for more than a decade. Though he and his team knew it was important to cellular activity, they didn't have a particular disease connection in mind. Together with colleagues in Japan, they did demonstrate its importance to autophagy – a process whose importance to disease research continues to grow as scientists learn more about it.

Several years ago, Guan's team stumbled upon clues that FIP200 might be important in neural stem cells when studying an entirely different phenomenon. They were using FIP200-less mice as comparisons in a study, when an observant postdoctoral fellow noticed that the mice experienced rapid shrinkage of the brain regions where neural stem cells reside.

"That effect was more interesting than what we were actually intending to study," says Guan, as it suggested that without FIP200, something was causing damage to the home of neural stem cells that normally replace nerve cells during injury or aging.

In 2010, they worked with other U-M scientists to show FIP200's importance to another type of stem cell, those that generate blood cells. In that case, deleting the gene that encodes FIP200 leads to an increased proliferation and ultimate depletion of such cells, called hematopoietic stem cells.

But with neural stem cells, they report in the new paper, deleting the FIP200 gene led neural stem cells to die and ROS levels to rise. Only by giving the mice the antioxidant n-acetylcysteine could the scientists counteract the effects.

"It's clear that autophagy is going to be important in various types of stem cells," says Guan, pointing to the new paper in Autophagy that lays out what's currently known about the process in hematopoietic, neural, cancer, cardiac and mesenchymal (bone and connective tissue) stem cells.

Guan's own research is now exploring the downstream effects of defects in neural stem cell autophagy – for instance, how communication between neural stem cells and their niches suffers. The team is also looking at the role of autophagy in breast cancer stem cells, because of intriguing findings about the impact of FIP200 deletion on the activity of the p53 tumour suppressor gene, which is important in breast and other types of cancer. In addition, they will study the importance of p53 and p62, another key protein component for autophagy, to neural stem cell self-renewal and differentiation, in relation to FIP200.

Friday, 5 April 2013

Scientists have shed light on a common bleeding disorder by growing and analysing stem cells from patients' blood to discover the cause of the disease in individual patients.

The technique may enable doctors to prescribe more effective treatments according to the defects identified in patients' cells.

In future, this approach could go much further: these same cells could be grown, manipulated, and applied as treatments for diseases of the heart, blood and circulation, including heart attacks and haemophilia.

The study focused on von Willebrand disease (vWD), which is estimated to affect 1 in 100 people and can cause excessive, sometimes life-threatening bleeding. vWD is caused by a deficiency of von Willebrand factor (vWF), a blood component involved in making blood clot. vWF is produced by endothelial cells, which line the inside of every blood vessel in our body. Unfortunately, they are difficult to study because taking biopsies from patients is invasive and unpleasant.

A group led by Dr Anna Randi at the National Heart and Lung Institute,Imperial College Londonused a new approach to investigate the disease. Dr Richard Starke, a British Heart Foundation Intermediate Fellow and lead author of the study, took routine blood samples from eight patients with vWD, extracted stem cells called endothelial progenitor cells, and grew them in the lab to yield large numbers of endothelial cells.

By testing these cells, they were able to analyse each patient's disease in unprecedented detail. In some patients, the scientists found new types of defect, which may enable them to recommend improved treatments. Professor Mike Laffan, a collaborator in the study and in charge of patients with VWD at Hammersmith Hospital in West London, is looking to apply these findings to reduce severe bleeding in these patients.

Dr Randi believes that endothelial progenitor cells could become an invaluable resource for testing new drugs for vWD and other diseases.

"We will be able to test the effects of a range of compounds in the patients' own cells, before giving the drugs to the patients themselves," she said.

This approach could have impact far beyond vWD. Endothelial cells derived from blood could also be isolated and reinjected into someone recovering from a heart attack, to help them grow new blood vessels and repair the injured heart tissue. Dr Starke says this approach avoids the main problem with transplant therapies, in which the immune system tries to destroy the foreign material.

"The patients would receive their own cells, so they wouldn't face the problems of rejection," he said.

Work is well underway towards achieving this goal, but blood-derived endothelial cells are only now being explored.

"There are already many studies where patients have been injected with stem cells to see whether damage to the heart could be repaired, and there are some promising results," says Dr Randi.

"The door is open to such treatments, and our studies are a step towards identifying the right cells to use."

The group's previous research has already thrown up pointers for potential new treatments. Aside from producing vWF to form clots, endothelial cells are responsible for forming new blood vessels. In their last paper, the group showed that vWF is actually needed to build healthy blood vessels. Some patients with vWD suffer severe bleeding from the gut because defects in vWF cause their blood vessels to develop abnormally.

"There are drugs already being used in other diseases which target abnormal blood vessel, that could be useful to stop bleeding in some vWD patients," says Randi.

"Nobody would have thought of using them to treat vWD, but by testing them on the patient's own endothelial cells, in the laboratory, we can find out if these drugs work before giving them to the patient."

Scientists are now interested in the possibility of using endothelial cells as a treatment in themselves. For instance, haemophilia, the hereditary bleeding disorder which affected Queen Victoria's family, might one day be treated by taking these cells from a patient and replacing the gene that causes the disease, then putting them back into the patient.